| Background:Dendritic Cell(DC) is widely recognized as the most efficient and professional antigen-presenting cells, which plays a crucial role in activating a specific immune response and its regulation. DC is mainly differentiated from the myeloid precursor, Bone marrow mononuclear cells(BMMC), of the pluripotent stem cells in bone marrow. During the process of immune response, DC selectively initiates immune response between specific immune reaction and immune tolerance by adjusting its expression of positive or negative costimulatory molecules and adhesion molecules(namely, Costimulatory Signals), exactly.PD-1/PD-L1(Program Death 1, PD-1; Program Death Ligand 1, PD-L1), one of the momentous negative costimulatory signaling pathway in the peripheral immune tolerance, plays a significant role in graft rejection of organ transplantation and immune regulation of tolerability. In this study, we developed a cytokine-induced "cocktail" training method to induce Myeloid-derived DC(MDDC) to highly express the negative costimulatory molecule PD-L1, namely PD-L1 highDC, then identified its morphological, molecular phenotype and functional characterization. Meanwhile, we explored the most suitable revulsive conditions for the expression of PD-L1 in vitro and built an efficient and practical immunological tolerance DC. Although the culture method of MDDC is relatively mature, there is little research about how to effectively induce and acquire PD-L1 highDC at home and abroad. Therefore, we tried to explore a cytokine-induced "cocktail" training method for PD-L1 highDC in vitro, and proceeded steady precendent experimental work for the immune tolerance research of organ transplantation in vivo.Objective: To explore a sententious and effective culture method for PD-L1 highDC induction which provided the extraneous experiment precondition for the mechanism study of tolerance regulation via PD-L1/PD-1 negative costimulatory signaling pathway by PD-L1 highDC reinfusion in liver transplantation.Methods:1. Experimental groups were presented as follow: Group induced by GM-CSF(Group GM-CSF), classic DC Group(Group c DC) and "cocktail" training Group(Group "cocktail"). Bone marrow cells(BMC) was obtained by aseptic technique and bone marrow mononuclear cells(BMMC) were purified by density gradient centrifugation from BMC, then BMMC were cultured in "cocktail" culture method adding cytokines GM-CSF, IL- 4 and IFN-γ for PD-L1 highDC training and ripened them with lipopolysaccharide(LPS) at eighth day.2. During the PD-L1 highDC training, growth state of PD-L1 highDC was observed by Inverted Microscope and its morphological features were recorded by Phase Contrast Microscope and Scanning Electron Microscope. The mature DC was collected for surface molecules(CD11c, CD86, MHC-Ⅱ, PD-L1) identification by Flow Cytometry and its biological function was detected by allogeneic Mixed Lymphocyte Reaction(MLR) as well.Result:1. Each method could be implemented to obtain typical DCs successfully and all DCs shared the similar morphology. The yield and purity of DC in Group c DC and Group "cocktail" were more than 80% and concentration of IFN-γ at 10ng/ml was optimal to harvest purified PD-L1 highDC.2. Surface molecules identification by Flow Cytometry displayed: the expression of PD-L1 in Group "cocktail"(82.26 ± 6.43) % was significant higher than that in Group c DC(2.00 ± 0.36)%, while MHC-Ⅱ, the main vehicle of antigen uptaking on DC, showed significant lower on PD-L1highDC(2.00 ± 0.36) % compared with c DC( 82.26 ± 6.43) %.3. MLR displayed: PD-L1 highDC showed a depressed capacity to stimulate allogeneic lymphocytes to proliferate, compared with c DC, and there was a statistical difference between the two groups(P <0.05).Conclusion:1. "Cocktail" training method was successful to induce and harvest a large amount of PD-L1 highDC which showed no significant difference in morphology, compared with classic DC.2. On the culture condition of "cocktail" training, the concentration of IFN-γ at 10ng/m L was optimal for the induction and training of high-purity PD-L1 highDC.3. Compared with classic DC, PD-L1 highDC showed a significantly feeble ability to stimulate allogeneic lymphocytes to proliferate which confirmed its suppressive biological characteristics.4. According to the significant statistical difference in the MHC-Ⅱ expression between c DC and PD-L1 highDC, we speculated that down-regulation of MHC-Ⅱ is one of the crucial causes leading to a feeble ability for stimulating allogeneic lymphocyte proliferation by PD-L1 highDC.5. This experiment successfully set up a cytokine-induced "cocktail" training method for PD-L1 highDC in vitro, which proceeded steady precendent experimental work for the immune tolerance research of organ transplantation... |
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